The effects of purified rhamnolipids from Pseudomonas aeruginosa on short circuit current in respiratory epithelium have been studied in sheep tracheal epithelium mounted in Ussing chambers under short circuit conditions. In low concentrations (100 microM) mucosal addition of rhamnolipids produces a decrease in short circuit current of 22% and in conductance of 4%. At higher concentrations (> 100 microM), large increases in tissue conductance accompany a greater reduction in short circuit current, suggesting disruption of the intercellular junctions. Serosal addition of rhamnolipids has no effect on ion transport. Pretreatment of the tissues with the sodium channel blocker amiloride (100 microM) or bathing the mucosal surface with sodium-free solution significantly decreased the rhamnolipid-induced fall in short circuit current but did not prevent it completely. Inhibition of chloride transport, sodium-glucose cotransport, and bicarbonate secretion with bumetanide, phloridzin, and acetazolamide, respectively, did not significantly alter the rhamnolipid effect. This suggests that the effect of rhamnolipids on short circuit current is mediated predominantly but not exclusively by an effect on sodium transport. The effects of rhamnolipids on ion transport occur at concentrations within the range occurring in the sputum of patients with cystic fibrosis. The changes in ion transport may explain some of the known effects of rhamnolipid on mucociliary clearance.